Project Summary/Abstract Parental history of type 2 diabetes (T2D), undernutrition, or obesity confers substantial risk for obesity and T2D in the offspring, with effects sustained over multiple generations. Genetic variation explains only a portion of this risk. Epigenetic mechanisms (e.g. DNA methylation, histone modification, noncoding RNA [ncRNA]), can regulate transcriptional responses to physiologic stimuli, independent of DNA sequence and can be maintained post-mitotically, imparting long-term effects. Maternal health during pregnancy is now recognized to shape epigenetic marks and disease phenotypes in offspring. However recent data from our lab and others show that health and nutrition of fathers, too, can contribute to disease risk in their progeny. We have shown that F1 offspring of females undernourished during pregnancy develop glucose intolerance, dysregulated lipid metabolism, and obesity ? similar to metabolic syndrome in humans. Strikingly, metabolic risk can also be transmitted to subsequent generations via the paternal lineage, yielding obesity and glucose intolerance in both F2 and F3 generations. Such paternal-lineage effects suggest that sperm or seminal fluid components may influence offspring development via epigenetic mechanisms stemming from prior environmental exposures. Supporting this possibility, we have shown that in utero undernutrition alters DNA methylation in spermatozoa of F1 offspring males in adulthood and expression of genes neighboring these loci in F2 embryos. Other studies have demonstrated modulation of sperm DNA methylation and ncRNA by diet, obesity, and diabetes. Together, these data support the hypothesis that metabolic health of males can alter sperm epigenetic marks, and suggest a novel, modifiable mechanism of transmission. Given the unprecedented T2D and obesity epidemics, and high risk of T2D in offspring of fathers with T2D, paternal diabetes is an important target for translational studies. However, it is unknown how human sperm epigenetic patterns change with glycemic control, and whether this may modify health of offspring. We hypothesize that epigenetic marks in human sperm are modified by a man's diabetes control. The Joslin WhyWAIT program, an intensive outpatient medical diabetes management program, has been shown to robustly improve glycemic control. Thus, we propose to test whether non-coding RNA and DNA methylation in human sperm can be modified in response to this intensive metabolic health intervention. In Aim 1, we will test whether improvements in glycemic control in men with T2D alter sperm DNA methylation and ncRNA species. In Aim 2, we will determine the background variation in the human sperm epigenome by analyzing epigenetic marks in men with and without T2D not undergoing metabolic intervention. In Aim 3, we will test causal relationships between glycemic control, BMI, ncRNA, and sperm DNA methylation using detailed bioinformatics and causal inference analysis. Together, these studies will provide proof-of-concept for modifiable mechanisms of paternal risk transmission and lay the foundation for future efforts to establish impact on offspring.